[go: up one dir, main page]

EP3020816A1 - Thérapie génique à médiation bactérienne - Google Patents

Thérapie génique à médiation bactérienne Download PDF

Info

Publication number
EP3020816A1
EP3020816A1 EP14192686.5A EP14192686A EP3020816A1 EP 3020816 A1 EP3020816 A1 EP 3020816A1 EP 14192686 A EP14192686 A EP 14192686A EP 3020816 A1 EP3020816 A1 EP 3020816A1
Authority
EP
European Patent Office
Prior art keywords
disease
cancer
cells
cell
live non
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP14192686.5A
Other languages
German (de)
English (en)
Inventor
Mark Tangney
William Byrne
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University College Cork
Original Assignee
University College Cork
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University College Cork filed Critical University College Cork
Priority to EP14192686.5A priority Critical patent/EP3020816A1/fr
Publication of EP3020816A1 publication Critical patent/EP3020816A1/fr
Withdrawn legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/1101—IkappaB kinase (2.7.11.10)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag

Definitions

  • Phagocytic cells such as macrophages, dendritic cells (DCs) and neutrophils fulfil essential roles in homeostasis, tissue repair and immunity. However they are also known to play a negative role, either aetiologically or pathophysiologically, in several diseases. Elevated circulating levels along with enhanced recruitment of such cells to inflamed tissues has been observed in a number of chronic inflammatory conditions including inflammatory bowel disease (IBD), rheumatoid arthritis (RA) and multiple sclerosis (MS). In active IBD, neutrophil accumulation in the intestinal lumen correlates with epithelial injury and clinical disease activity. Importantly manipulating phagocytic cells has proven therapeutically beneficial; neutrophil depletion provided promising results in IBD clinical trials. Similarly, targeting immunogenic DCs by blocking their persistent activation of self-reactive T cells with a monoclonal antibody yielded positive clinical benefits in patients with RA, IBD and psoriasis.
  • IBD inflammatory bowel disease
  • phagocytic cells are often especially problematic. Macrophages perpetuate a cycle of chronic inflammation that provokes cancer initiation. They facilitate angiogenesis, tumour cell migration, invasion and intravasation, all leading to metastatic disease. Strategies to manipulate tumour-associated macrophages have shown promise in pre-clinical cancer models and in clinical trials.
  • tumour associated neutrophils TANs
  • Pro-tumoural functions of TANs have been reported to include angiogenesis, blockade of anti-tumoural immune responses and promotion of tumour cell proliferation, invasion and spread.
  • Tumour-promoting DCs have been identified in both breast and pancreatic cancer. DC maturation is subverted by the tumour which skews the immune response towards a Th2 phenotype and production of tumour- promoting cytokines.
  • Hagemann 1 first proposed a strategy and validated the therapeutic effect of treating ovarian cancer by re-educating ascitic macrophages.
  • the Hagemann study isolated TAM from the ascitic fluid of mice, grew them in the lab, placed the gene in the cells ex vivo (utilising traditional chemical methods), and then introduced these newly re-educated cells into mice.
  • the therapeutic effects were very powerful.
  • this ex vivo method of transfecting TAM is very artificial and impractical/expensive in a clinical setting, precluding commercial/clinical exploitation of the strategy.
  • the invention addresses at least one of the problems of the prior art by providing gene therapy of diseases characterised by macrophage accumulation (i.e. solid tumor cancer and malignant ascites) in which a non-invasive bacterium is employed as a delivery vehicle.
  • a non-invasive bacterium as a delivery vehicle selectively targets the transgene to disease-associated macrophage (and other phagocytic cells), where the transgene is expressed by the non-invasive bacterium or passively transfects the macrophage.
  • the transgene typically encodes an effector molecule capable of re-programming the disease associated macrophage from a pro-disease phenotype to an anti-disease phenotype, or an effector molecule that directly targets disease cells to attenuate or inhibit the disease pathology.
  • an in vitro -differentiated human monocyte cell line and two in vivo mouse models proof of delivery is demonstrated with bacteria carrying reporter constructs and therapeutic constructs.
  • the data demonstrate the ability to specifically transfect (deliver genes to) phagocytic cells within the malignant peritoneum in a mouse model, the ability to specifically transfect (deliver genes to) phagocytic cells within solid tumours in a mouse model, and demonstrates an improvement in the immune environment at the cancer locus, as a result of delivery of a therapeutic gene in subcutaneous tumour mouse models.
  • the invention provides a live non-invasive bacterium comprising a transgene, typically under the control of a promotor, wherein the transgene encodes a phagocytic cell-relevant therapeutically active agent.
  • the transgene encodes a tumor associated macrophage (TAM) relevant therapeutically active agent.
  • TAM tumor associated macrophage
  • the phagocytic cell relevant therapeutically active agent comprises an effector molecule capable of re-programming a disease-associated phagocytic cell from a pro-disease phenotype to an anti-disease phenotype.
  • the phagocytic cell relevant therapeutically active agent comprises an effector molecule capable of re-programming a disease-associated macrophage cell from a pro-disease phenotype to an anti-disease phenotype.
  • the phagocytic cell relevant therapeutically active agent comprises an effector molecule capable of re-programming a tumor-associated macrophage cell from a pro-disease phenotype to an anti-disease phenotype.
  • the effector molecule comprises a macrophage NF ⁇ B pathway attenuator.
  • the effector molecule comprises a mediator that modulates host cell cytokine profile (for example by inducing a pro-inflammatory response (in the case of treatment of cancer) or inhibiting a pro-inflammatory response (in the case of an inflammatory disease such as cardiovascular disease or rheumatoid arthritis)).
  • the effector molecule comprises a mediator that induces expression of a pro-inflammatory cytokine (for example IL-1, IFN ⁇ or TNF ⁇ .
  • the effector molecule comprises a mediator that inhibits expression of a pro-inflammatory cytokine (for example IL-1 or TNF ⁇ ).
  • the tumor associated macrophage (TAM) relevant therapeutically active agent comprises an effector molecule capable of directly targeting a cancer cell.
  • the effector molecule is a monoclonal antibody.
  • the promotor is configured to mediate expression in a eukaryotic cell.
  • the promotor is a eukaryotic promotor.
  • the therapeutically active agent is selected from a protein, a peptide, an oligopeptide, a polypeptide, and a low molecular weight gene expression inhibitor or activator.
  • the low molecular weight gene expression inhibitor or activator is a nucleic acid, preferably an RNA molecule. Examples include siRNA, shRNA, saRNA, miRNA or a ribozyme.
  • the effector molecule capable of re-programming a disease associated macrophage from a pro-disease phenotype to an anti-disease phenotype is a low molecular weight gene expression inhibitor or activator, preferably an RNA molecule.
  • the effector molecule capable of re-programming a tumor associated macrophage (TAM) from a pro-disease phenotype to an anti-disease phenotype is a low molecular weight gene expression inhibitor or activator, preferably an RNA molecule.
  • the invention also provides a live non-invasive bacterium according to the invention, for use in a method of treating a disease in a mammal characterised by phagocytic cell accumulation at a disease locus in the mammal, wherein the phagocytic cell-relevant therapeutically active agent is specific for the disease.
  • the invention also relates to a method of treating a disease in a mammal characterised by phagocytic cell accumulation at a disease locus in the mammal comprising a step of administering to the mammal a live non-invasive bacterium according to the invention, wherein the phagocytic cell-relevant therapeutically active agent is specific for the disease.
  • the live non-invasive bacterium according to the invention is administered locally to a disease locus in the mammal.
  • the disease is cancer.
  • the disease is selected from a solid tumor and malignant ascites.
  • the disease is cancer
  • the phagocytic cell-relevant therapeutically active agent comprises an effector molecule capable of re-programming a tumor associated macrophage (TAM) from a pro-tumorigenic phenotype to an anti-tumor phenotype.
  • TAM tumor associated macrophage
  • the effector molecule is a low molecular weight inhibitor of macrophage NF ⁇ B pathway or a modulator of host cell cytokine expression.
  • the disease is cancer
  • the phagocytic cell-specific therapeutically active agent comprises an effector molecule capable of directly targeting a cancer cell.
  • the effector molecule is a protein or peptide drug, for example monoclonal antibody.
  • the cancer is a solid tumor cancer, and in which the live non-invasive bacteria is administered intratumorally.
  • the cancer is a solid tumor cancer, and in which the live non-invasive bacteria is administered intravenously.
  • the cancer is a peritoneal cancer, and in which the live non-invasive bacteria is administered intraperitoneally.
  • Balb/C mice bearing s.c. CT26 tumours were treated with PBS, E.coli or E.coli + IKK2-DN plasmid.
  • the cytokine profile within the tumour was determined using a mouse pro-inflammatory multiplex plate. Levels are expressed as the mean +/- SEM for 3 mice per group.
  • the left panel displays individual cytokine profiles at three distinct time points post bacterial administration.
  • the right panel focuses on Day four where the greatest change in cytokine levels was detected.
  • Statistics compare the two bacterial treated groups where one group received the therapeutic IKK2-DN gene and the other did not. *indicates p ⁇ 0.05. **indicates p ⁇ 0.005.
  • Non-invasive bacterium means a bacterium that cannot actively mediate its internalisation to a eukaryotic cell, lacks invasion factors, and is non-pathogenic and non-virulent, but is capable of being taken up by a phagocytic cell. Examples of non-invasive bacteria include non-pathogenic strains of E.
  • Non-pathogenic Lactic Acid Bacteria such as bifidobacteria, lactobacilli and non-pathogenic Gram positive cocci such as non-pathogenic members of the genera Streptoccocus and Lactococcus; Non-pathogenic members of Gram-positive genera such as Listeria (e.g. L. welshimeri); Strains of Gram negative or Gram positive bacteria rendered non-invasive by genetic modification (e.g. species of Salmonella rendered non-invasive by deletion of relevant virulance factors).
  • Transgene means an exogenous gene or genetic material that is transferred into the non-invasive bacteria by means of a genetic engineering technique, and which is capable of being expressed by the non-invasive bacteria or a phagocytic cell targeted by the non-invasive bacteria.
  • transgenes includes nucleic acids encoding for proteins, peptides, oligopeptides, polypeptides, mRNA capable of interfering with host cell transcription, low molecular weight inhibitors of gene expression, including siRNA, shRNA, miRNA, ribozymes, or activators of gene expression such as small activating RNA (saRNA).
  • transgene and promotor are provided in the form of a suitable vector, for example a plasmid comprising the transgene and the promotor.
  • suitable vectors include any plasmid capable of being maintained by the relevant bacterial cell (e.g. features a suitable origin of replication, such as of the pUC, pAMbeta type etc.) with the capacity to introduce and maintain heterologous DNA sequences within the plasmid (such as within a multiple cloning site).
  • the heterologous DNA may feature a promotor suitable for mediating expression of the sequence relevant to the therapeutic strategy (see transgene).
  • Promotor means a nucleic acid operably connected to a transgene and capable of driving expression of the transgene in either the bacteria or a host phagocytic cell.
  • the promotor may be a prokaryotic promotor (a promotor capable of driving expression of the transgene using the genetic machinery of the non-invasive bacteria) or a eukaryotic promotor (a promotor capable of driving expression of the transgene using the genetic machinery of a eukaryotic host cell).
  • Phagocytic cell-relevant as applied to a therapeutically active agent means an agent that is therapeutically active against a disease characterised by phagocytic cell accumulation at the site of the disease.
  • diseases i.e. "phagocytic cell-relevant diseases”
  • cancer especially solid tumors and malignant ascites
  • cardiovascular disease a phagocytic cell relevant disease
  • rheumatoid arthritis all of which are characterised by macrophage accumulation at a disease locus.
  • the phagocytic cell-relevant disease is a macrophage cell relevant disease.
  • Therapeutically active agent means an agent that when administered to a mammal exhibiting a phagocytic cell-relevant disease is capable of attenuating or inhibiting disease pathology or progression by means of (a) directly targeting the disease cells to attenuate or inhibit disease pathology (for example a therapeutic monoclonal antibody that targets cancer cells), or (b) re-programming a phagocytic cell (typically a macrophage) from a pro-disease phenotype to an anti-disease phenotype (examples include inhibiton of the macrophage NF ⁇ B pathway, or a mediator that modulates host cell cytokine profile).
  • the phagocytic cell-specific therapeutically active agent is a macrophage-specific therapeutically active agent.
  • Non-immunogenic as applied to a therapeutically active agent means a therapeutically active agent that does not. present to the host immune system as an antigen.
  • Tumor associated macrophage (TAM) relevant therapeutically active agent means an agent that when administered to a mammal with a cancer characterised by accumulation of macrophages at a cancer locus is capable of attenuating or inhibiting disease pathology or progression by means of (a) directly targeting the cancer cells to attenuate or inhibit disease pathology or progression (for example a therapeutic monoclonal antibody that targets cancer cells), or (b) re-programming a tumor-associated macrophage from a pro-disease phenotype to an anti-disease phenotype (examples include an inhibitor of the macrophage NF ⁇ B pathway, or a mediator that modulates host cell cytokine profile).
  • “Therapy” or “treatment” should be taken to mean a course of action / dosing regime that either inhibits, delays or prevents the progression of disease.
  • Phagocytic cell means a cell that protects the body by ingesting foreign matter, and includes white blood cells such as neutrophils, monocytes, macrophages and mast cells, and dendritic cells.
  • white blood cells such as neutrophils, monocytes, macrophages and mast cells, and dendritic cells.
  • the phagocytic cell is a macrophage.
  • Disease-associated macrophage means a macrophage that accumulates at a disease locus, and would include cancer-associated macrophage, coronary disease associated macrophage, and arthritis-associated macrophage.
  • cancer associated macrophage include tumor-associated macrophage (TAM) that are known to accumulate in cancer tumors, especially solid tomors, and cancer cell associated macrophage (for example macrophage that accumulate at a site of malignant ascites).
  • TAM tumor-associated macrophage
  • cancer cell associated macrophage for example macrophage that accumulate at a site of malignant ascites.
  • Pro-disease phenotype as applied to a disease-associated macrophage means a phenotype that contributes to the disease pathology, generally by means of modulating the macrophages' immune response to the disease.
  • Tumor-associated macrophage TAMs
  • M2 Tumor-associated macrophage
  • inflammatory diseases such as arthritis, associated with excessive local pro-inflammatory macrophage activity (of the "M1" phenotype) play a detrimental role in disease pathophysiology.
  • Anti-disease phenotype as applied to a disease-associated macrophage means a phenotype that attenuates or inhibits the pathology of the disease, generally by means of modulating the macrophages' immune response to the disease.
  • the anti-disease phenotype means that the macrophages pro-disease phenotype is attenuated or inhibited.
  • the phenotype of the TAM is modified such that tolerance to tumor cells is switched off, or that the macrophage mediates an anti-tumor immune response.
  • Effective molecule means a molecule that is capable of re-programming a disease associated macrophage from a pro-disease phenotype to an anti-disease phenotype.
  • Example of such molecules are proteins, peptides (including oligopeptides and polypeptides), and low molecular weight inhibitors of expression of specific genes.
  • the effector molecule may be selected from a therapeutic antibody, a cytokine, or an enzyme inhibitor therapeutic.
  • Therapeutic antibody means an antibody, generally a monoclonal antibody, that has a therapeutic effect in a mammal.
  • therapeutic antibodies indicated for cancer include Catumaxomab (indicated for treatment of malignant ascites), and Bevacizumab (indicated for treatment of metastatic cancers).
  • Enzyme inhibitor therapeutic means an enzyme inhibitor that is indicated for the treatment of one or more cancers.
  • examples include tyrosine kinase inhibitors (TKI) for example Sunitinib (indicated for renal cell carcinoma), Imatinib (indicated for leukaemia), Gefitinib (indicated for certain breast and lung cancers), and Erlotinib (indicated for non-small cell lung carcinoma and pancreatic cancer).
  • TKI tyrosine kinase inhibitors
  • Cytokine refers to a small protein involved in cell signalling and includes chemokines, interferons, interleukins, tumor necrosis factors.
  • Low molecular weight inhibitor refers to a nucleic acid molecule configured to inhibit expression of a specific target gene. Examples include siRNA, shRNA, miRNA, antisense oligonucleotides, and ribozyme molecules.
  • Small inhibitory RNA are small double stranded RNA molecules which induce the degradation of mRNAs.
  • Micro RNA's are single stranded ( ⁇ 22nt) non-coding RNAs (ncRNAs) that regulate gene expression at the level of translation.
  • small hairpin RNA (shRNA) molecules are short RNA molecules having a small hairpin loop in their tertiary structure that may be employed to silence genes.
  • miRNA or shRNA molecules capable of silencing a specific gene will be apparent to those skilled in the field of miRNA or shRNA molecule design.
  • the level of expression of a specific target gene can be modulated using antisense or ribozyme approaches to inhibit or prevent translation of mRNA transcripts or triple helix approaches to inhibit transcription of the specific gene.
  • Antisense approaches involve the design of oligonucleotides (either DNA or RNA) that are complementary to mRNA for the gene. The antisense oligonucleotides will bind to the complementary mRNA transcripts and prevent translation.
  • Ribozyme molecules designed to catalytically cleave mRNA transcripts from a specific gene can also be used to prevent translation and expression.
  • RNA small activating RNA
  • the therapeutically active agent has therapeutic activity against the disease.
  • the therapeutically active agent may be an anti-cancer drug capable of being expressed by a transformed phagocytic cell (for example an anti-cancer peptide or monoclonal antibody), or an agent capable of re-programming the phagocytic cell to anti-cancer phenotype (for example by inhibition of the NF ⁇ B pathway.
  • the cancer is selected from the group comprising: esophagogastric cancer; fibrosarcoma; myxosarcoma; liposarcoma; chondrosarcoma; osteogenic sarcoma; chordoma; angiosarcoma; endotheliosarcoma; lymphangiosarcoma; lymphangioendotheliosarcoma; synovioma; mesothelioma; Ewing's tumor; leiomyosarcoma; rhabdomyosarcoma; colon carcinoma; colorectal carcinoma; pancreatic cancer; breast cancer; ovarian cancer; prostate cancer; squamous cell carcinoma; basal cell carcinoma; adenocarcinoma; sweat gland carcinoma; sebaceous gland carcinoma; papillary carcinoma; papillary adenocarcinomas; cystadenocarcinoma; medullary carcinoma; bronchogenic carcinoma; renal cell carcinoma; hepatoma; bile duct carcinoma
  • E.coli K12 MG1655 was used in all experiments (UCC culture collection).
  • a GFP plasmid (pMAX-GFP, Amaxa, USA) under the control of a eukaryotic promoter was introduced into this strain by electro-transformation.
  • the hlyA gene was introduced into bacteria also 2 . Where relevant, heat-killing of the bacteria was performed by heating at 95° C for 1 hour.
  • E. coli a hlyA -expressing E. coli MG1655 strain (referred to as E. coli hereafter) was employed; MG1655 was transformed with a hlyA plasmid (pNZ44 - generated by Dr. Joanne Cummins, Cork Cancer Research Centre).
  • the mCherry-bearing plasmid (Takara Bio Europe/Clontech, France) is commercially available.
  • a plasmid bearing a FLAG-tagged protein (a kinase-negative mutant of IKK2) was a kind gift from Dr. Rainer de Martin 3 .
  • E. coli was transformed with the therapeutic plasmid carrying a kinase-negative mutant of IKK2 (IKK2-DN) - a kind gift from Dr. Rainer de Martin 7 .
  • GFP and IKK2-DN genes were driven by eukaryotic promoters. Presence of the hlyA and therapeutic plasmid in the bacteria was confirmed by colony PCR to detect the hlyA and ampicillin resistance genes respectively.
  • the appropriate bacterial stock was inoculated into 10 ml LB-broth with antibiotic selection and grown overnight ( ⁇ 16 H) at 37°C, shaking at 200 rpm.
  • the culture was grown for a further 2-3 H at 37°C, shaking at 200 rpm and the bacteria harvested when the OD 600 reached 0.6-0.8.
  • 1 ml culture was then pelleted by centrifugation (13500 rpm, 1 min), the pellet was washed twice in PBS and resuspended in 1 ml PBS. The concentration of this solution had previously been determined to be approximately 1x10 9 CFU/ml. This stock solution was then diluted in PBS to the appropriate concentration.
  • RAW 264.7 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and maintained in DMEM medium (Sigma-Aldrich) supplemented with 10% foetal bovine serum (FBS) (Sigma-Aldrich) in a 37°C incubator with 5% CO 2 .
  • FBS foetal bovine serum
  • 6 x10 5 cells were seeded per well of a 6-well plate in 2ml media. Cell adherence and a spreading morphology were confirmed using a light microscope. After 24 H the media was changed before the cells were used for further experimentation.
  • Bacteria carrying pMAX-GFP were prepared as above and added directly to the RAW 264.7 macrophages at different multiplicities of infection (MOI).
  • a cytotoxicity assay was performed to assess cell viability in response to each of the experimental conditions using the Nucleocounter system (ChemoMetec, Bioimages Ltd). Cells were resuspended in PBS with 10% FBS to prevent clumping prior to analysis. A FACSLSRII 5 laser (UV/violet/blue/yellow-green/red) cytometer and BD Diva software (Becton, Dickinson) were used to measure GFP positivity. For each sample, 50-100,000 events were recorded. Dead cells/debris were gated out of the analysis based on their low FSC and high SSC characteristics and autofluorescence was controlled by the appropriate gating controls. The proven gene transfer vector Lipofectamine 2000 (Invitrogen) was used as a positive control.
  • the ID-8-Fluc cell line was a kind gift from Dr. Kah Whye Peng, Mayo Clinic, USA.
  • 6-8 week old albino C57BL/6 mice Hard, UK
  • i.p. intraperitoneally
  • 1 x 10 6 luciferase-expressing mouse ovarian surface epithelial ID8 cells were injected directly into the peritoneum leading to the formation of multiple tumours and ascites within 8 weeks (data not shown).
  • mice All mice were housed in a conventional environment (temperature 21°C, 12 h light: 12 h darkness, humidity 50%) in a dedicated animal holding facility. They were fed a standard non-sterile pellet diet and tap water ad libitum. Mice were allowed 2 weeks to acclimatize before entering the study. All animal procedures were performed according to national ethical guidelines following approval by the University College Cork Animal Experimentation Ethics Committee.
  • a plasmid bearing a FLAG-tagged protein (IKK2-DN) was used as a reporter for detection of gene delivery within solid CT26 tumours.
  • the cells were pelleted by centrifugation (1500 rpm for 5 min at 4°C), washed twice in PBS and resuspended in 2 ml red cell lysis buffer (Sigma) with 5 min incubation at room temperature. 30 ml media with serum was added to stop the lysis process. Cells were recovered by centrifugation again, washed once in PBS and counted - both viable and non-viable. 1x10 6 viable cells were aliquoted per sample to be analysed and cells were fixed and stained.
  • anti-F4/80-PE clone BM8
  • anti-CD68-PerCP/Cy5.5 clone FA-11
  • anti-FLAG Alexa Fluor 647 Cell signalling, 1 in 450 dilution
  • tumour samples were harvested from animals, flash frozen and stored at -80°C. Cytokine analysis was performed on a mouse pro-inflammatory multi-plex plate (MSD) as per the manufacturer's instructions. The following preparatory work was performed on tumour samples; samples were thawed on ice and a 300 mg specimen was isolated. The appropriate volume of homogenisation buffer (PBS with protease inhibitor cocktail [Roche]) was added to each specimen to yield a concentration of 100 mg of tissue per ml and a uniform solution achieved using a tissue homogeniser. 500 ⁇ l of this solution was centrifuged at 1000g and the supernatant recovered. The supernatant was diluted 1 in 20 and 50 ⁇ l (equivalent to 250 ⁇ g of tissue) added per well.
  • PBS with protease inhibitor cocktail [Roche] protease inhibitor cocktail
  • TumCherry gene delivery was used to detect mCherry gene delivery to solid CT26 tumours. Tumours were removed, halved and snap-frozen in optimal cutting temperature compound (Tissue-Tek; Sakura Finetek) using liquid nitrogen. Frozen tumours were cryosectioned (5 ⁇ m), fixed for 5 min in ice-cold acetone-ethanol (3:1 ratio), and blocked with blocking serum for 45 min at room temperature in a humidified chamber. Blocked sections were stained with purified rat monoclonal F480 antibody (Clone: CI:A3-1, Abcam) and counterstained using goat anti-rat Alexa Fluor 488-conjugated anti-IgG antibody (Invitrogen, Biosciences Ltd).
  • Flow cytometry was used to detect mCherry gene delivery within ID8 ascites and to assess vector delivery selectivity and vector-mediated recruitment of phagocytic cells.
  • Approximately 1ml ascites fluid was harvested from the peritoneum of each animal via a 23- gauge needle and kept on ice until processing. Cells were recovered from the ascites by centrifugation at 1500rpm for 5 min, washed twice in PBS and subjected to red cell lysis buffer (Sigma) for 5 min. Media with serum was added to stop lysis. Cells were recovered by centrifugation again, washed in PBS and viable and non-viable cells were counted.
  • Dead cells/debris were gated out of the analysis based on their low FSC and high SSC characteristics. Background staining was controlled by the use of FMO (fluorescence minus one) controls. The results represent the percentage of positively stained cells in the total cell population exceeding the background staining signal.
  • the specificity of vector delivery was determined by gating on the mCherry + cells and identifying cell types using the following antibodies; anti-F4/80-PE (Clone: BM8, Biolegend), anti-CD11c-PE-CY7 (Clone: HL7, BD Pharmingen) and anti-LY6G/6C-APC-CY (Clone: RB6-8C5, Biolegend).
  • Macrophages were defined as F480 +
  • DCs were defined as CD11C +
  • neutrophils were defined as Ly6G +
  • T cells were defined as CD3 + and identified using anti-CD3 AF700 (Clone: eBio500A2, eBioscience) antibody
  • NK cells were defined as DX5 + and identified using anti-CD49b- Pe-CY7 (Clone DX5, Biolegend) and NK T cells were defined as CD3 + DX5 + .
  • E. coli MG1655 mediates gene delivery to phagocytic cells in vitro
  • GFP expression was measured only within the viable cell population as determined by their FSC/ SSC characteristics. Gating on the non-viable cell population yielded negligible GFP positivity. While lipofection resulted in detection of fewer transfected cells than bactofection at MOI 300, resulting transgene expression was much higher as evidenced by brighter fluorescence within lipofected cells on both FACS and microscopic analyses.
  • E. coli MG1655 mediates gene delivery to phagocytic cells in vivo
  • phagocytic cells accounted for 100% of mCherry + cells at both time points, indicating that this gene delivery strategy is specific for phagocytic cells ( Fig. 5a ).
  • macrophages represented the dominant phagocytic cell type scavenging the bacterial vector, with 96% of mCherry + cells expressing the macrophage marker F4/80, while the remaining 4% were either DCs or neutrophils.
  • reporter gene expression was similar between macrophages (33%), DCs (40%) and neutrophils (27%) ( Fig 5b ).
  • FMO controls are presented in Suppl.
  • Fig. 4 the identity of the mCherry + cells is based on strict appropriation of cell surface marker expression to definitive cell types and is open to interpretation given the high level of plasticity associated with phagocytic populations. Indeed, considerable overlap between surface markers was observed ( Fig. 5C ).
  • Bacterial delivery provokes phagocytic cell differentiation in ascites
  • the bacterial vector did not result in a change to the total number of cells per ml of ascites fluid recovered ( Fig. 6a ).
  • the percentage of phagocytic cells (identified as F4/80 + macrophages, CD11c + DCs or Ly6G + neutrophils) was increased at every time point following bacterial administration ( Fig. 6b and c). This suggested that immature phagocytic cells were differentiating in response to the bacterial vector.
  • macrophages were the predominant phagocytic cell representing 40% of the total ascitic cell population.
  • E.coli +/- IKK2-DN encoding plasmid, or PBS was i.t. injected to growing CT26 tumours.
  • the cytokine profile within the tumour was assessed at three time points post treatment. Results are displayed in Fig. 7 . Highest differences overall were observed four days post treatment, and statistics for this timepoint are displayed in Fig. 7 (right panel). At day four, significant change in cytokine levels could be attributed to the IKK2-DN gene. Cytokine changes invoked within the tumour involved significantly increased (p ⁇ 0.05) IL-12, IL-1 ⁇ , IL-6 and mKC, which in all cases is consistent with a pro-inflammatory response.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
EP14192686.5A 2014-11-11 2014-11-11 Thérapie génique à médiation bactérienne Withdrawn EP3020816A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP14192686.5A EP3020816A1 (fr) 2014-11-11 2014-11-11 Thérapie génique à médiation bactérienne

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
EP14192686.5A EP3020816A1 (fr) 2014-11-11 2014-11-11 Thérapie génique à médiation bactérienne

Publications (1)

Publication Number Publication Date
EP3020816A1 true EP3020816A1 (fr) 2016-05-18

Family

ID=51870914

Family Applications (1)

Application Number Title Priority Date Filing Date
EP14192686.5A Withdrawn EP3020816A1 (fr) 2014-11-11 2014-11-11 Thérapie génique à médiation bactérienne

Country Status (1)

Country Link
EP (1) EP3020816A1 (fr)

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017181148A2 (fr) 2016-04-15 2017-10-19 Alpine Immune Sciences, Inc. Protéines immunomodulatrices à variants du ligand icos et leurs utilisations
WO2017181152A2 (fr) 2016-04-15 2017-10-19 Alpine Immune Sciences, Inc. Protéines immunomodulatrices à variants de cd80 et leurs utilisations
WO2018022945A1 (fr) 2016-07-28 2018-02-01 Alpine Immune Sciences, Inc. Protéines immunomodulatrices à variants de cd112 et utilisations associées
WO2018022946A1 (fr) 2016-07-28 2018-02-01 Alpine Immune Sciences, Inc. Protéines immunomodulatrices à variants de cd155 et leurs utilisations
WO2018075978A1 (fr) 2016-10-20 2018-04-26 Alpine Immune Sciences, Inc. Protéines immunomodulatrices sécrétables de type variant et thérapie cellulaire utilisant des cellules obtenues par génie génétique
WO2018170026A2 (fr) 2017-03-16 2018-09-20 Alpine Immune Sciences, Inc. Protéines immunomodulatrices à variants de cd80 et leurs utilisations
WO2018170023A1 (fr) 2017-03-16 2018-09-20 Alpine Immune Sciences, Inc. Protéines immunomodulatrices à variants pd-l2 et utilisations associées
WO2018170021A1 (fr) 2017-03-16 2018-09-20 Alpine Immune Sciences, Inc. Protéines immunomodulatrices de variants de pd-l1 et utilisations associées
WO2019074983A1 (fr) 2017-10-10 2019-04-18 Alpine Immune Sciences, Inc. Protéines immunomodulatrices de variants de ctla-4 et leurs utilisations
WO2019079520A2 (fr) 2017-10-18 2019-04-25 Alpine Immune Sciences, Inc. Protéines immunomodulatrices à variants de ligand de icos variant, compositions et méthodes associées
WO2019136179A1 (fr) 2018-01-03 2019-07-11 Alpine Immune Sciences, Inc. Protéines immunomodulatrices multi-domaine et leurs méthodes d'utilisation
WO2019241758A1 (fr) 2018-06-15 2019-12-19 Alpine Immune Sciences, Inc. Protéines immunomodulatrices de variants de pd-l1 et utilisations associées
WO2020113141A2 (fr) 2018-11-30 2020-06-04 Alpine Immune Sciences, Inc. Protéines immunomodulatrices à variants cd86 et leurs utilisations
US11471488B2 (en) 2016-07-28 2022-10-18 Alpine Immune Sciences, Inc. CD155 variant immunomodulatory proteins and uses thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990011364A1 (fr) 1989-03-24 1990-10-04 University Patents, Inc. Procede et endoribonucleases de restriction de ribozymes d'arn
WO1997014806A2 (fr) * 1995-10-20 1997-04-24 Cambridge University Technical Services Limited Administration de polypeptides biologiquement actifs
WO2000009733A1 (fr) * 1998-08-13 2000-02-24 The Regents Of The University Of California Vecteurs d'apport intracellulaire
WO2009095436A2 (fr) * 2008-01-29 2009-08-06 Æterna Zentaris Gmbh Bactérie non pathogène et/ou atténuée capable d’induire l’apoptose dans des macrophages, procédé de fabrication de celle-ci et utilisations de celle-ci
WO2014198002A1 (fr) * 2013-06-14 2014-12-18 Ottawa Hospital Research Institute Bactérie produisant une protéine se liant à l'interféron et ses utilisations

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990011364A1 (fr) 1989-03-24 1990-10-04 University Patents, Inc. Procede et endoribonucleases de restriction de ribozymes d'arn
WO1997014806A2 (fr) * 1995-10-20 1997-04-24 Cambridge University Technical Services Limited Administration de polypeptides biologiquement actifs
WO2000009733A1 (fr) * 1998-08-13 2000-02-24 The Regents Of The University Of California Vecteurs d'apport intracellulaire
WO2009095436A2 (fr) * 2008-01-29 2009-08-06 Æterna Zentaris Gmbh Bactérie non pathogène et/ou atténuée capable d’induire l’apoptose dans des macrophages, procédé de fabrication de celle-ci et utilisations de celle-ci
WO2014198002A1 (fr) * 2013-06-14 2014-12-18 Ottawa Hospital Research Institute Bactérie produisant une protéine se liant à l'interféron et ses utilisations

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
BYRNE W L ET AL: "Bacterial-mediated DNA delivery to tumour associated phagocytic cells", JOURNAL OF CONTROLLED RELEASE, vol. 196, 17 November 2014 (2014-11-17), pages 384 - 393, XP029112421, ISSN: 0168-3659, DOI: 10.1016/J.JCONREL.2014.10.030 *
GUO H; ZHANG J; INAL C; NGUYEN T; FRUEHAUF JH; KEATES AC; LI CJ.: "Targeting tumor gene by shRNA-expressing Salmonella-mediated RNAi", GENE THERAPY, vol. 18, 2011, pages 95, XP055160971, DOI: doi:10.1038/gt.2010.112
HAGEMANN T ET AL: ""Re-educating" tumor-associated macrophages by targeting NF- B", ANNALS OF THE RHEUMATIC DISEASES, vol. 59, no. 90001, 1 June 2008 (2008-06-01), pages i54 - 1268, XP055189526, ISSN: 0003-4967, DOI: 10.1136/ard.59.suppl_1.i54 *
HAGEMANN T; LAWRENCE T; MCNEISH I; CHARLES KA; KULBE H; THOMPSON RG; BALKWILL FR: "Re-educating'' tumor-associated macrophages by targeting NF-kappaB", THE JOURNAL OF EXPERIMENTAL MEDICINE, vol. 205, 2008, pages 1261
KONO Y ET AL: "Antitumor effect of nuclear factor-[kappa]B decoy transfer by mannose-modified bubble lipoplex into macrophages in mouse malignant ascites", CANCER SCIENCE, vol. 105, no. 8, 1 August 2014 (2014-08-01), pages 1049 - 1055, XP055189514, ISSN: 1347-9032, DOI: 10.1111/cas.12452 *
LIZOTTE P H ET AL: "Attenuated Listeria monocytogenes reprograms M2-polarized tumor-associated macrophages in ovarian cancer leading to iNOS-mediated tumor cell lysis", ONCOIMMUNOLOGY, vol. 3, no. 5, 1 May 2014 (2014-05-01), pages e28926, XP055189509, DOI: 10.4161/onci.28926 *
OITZINGER W; HOFER-WARBINEK R; SCHMID JA; KOSHELNICK Y; BINDER BR; DE MARTIN R.: "Adenovirus-mediated expression of a mutant IkappaB kinase 2 inhibits the response of endothelial cells to inflammatory stimuli", BLOOD, vol. 97, 2001, pages 1611
PARSA S ET AL: "A high-throughput comparison of recombinant gene expression parameters for E. coli-mediated gene transfer to P388D1 macrophage cells", JOURNAL OF BIOTECHNOLOGY, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 137, no. 1-4, 10 October 2008 (2008-10-10), pages 59 - 64, XP025471507, ISSN: 0168-1656, [retrieved on 20080723], DOI: 10.1016/J.JBIOTEC.2008.07.1815 *
SARVER ET AL., SCIENCE, vol. 247, 1990, pages 1222 - 1225
ZURITA-TURK M ET AL: "Lactococcus lactis carrying the pValac DNA expression vector coding for IL-10 reduces inflammation in a murine model of experimental colitis", BMC BIOTECHNOLOGY, BIOMED CENTRAL LTD. LONDON, GB, vol. 14, no. 1, 9 August 2014 (2014-08-09), pages 73, XP021191870, ISSN: 1472-6750, DOI: 10.1186/1472-6750-14-73 *

Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11359022B2 (en) 2016-04-15 2022-06-14 Alpine Immune Sciences, Inc. CD80 variant immunomodulatory proteins and uses thereof
WO2017181152A2 (fr) 2016-04-15 2017-10-19 Alpine Immune Sciences, Inc. Protéines immunomodulatrices à variants de cd80 et leurs utilisations
US10882914B2 (en) 2016-04-15 2021-01-05 Alpine Immune Sciences, Inc. ICOS ligand variant immunomodulatory proteins and uses thereof
US11479609B2 (en) 2016-04-15 2022-10-25 Alpine Immune Sciences, Inc. CD80 variant immunomodulatory proteins and uses thereof
US11078282B2 (en) 2016-04-15 2021-08-03 Alpine Immune Sciences, Inc. CD80 variant immunomodulatory proteins and uses thereof
US11498967B2 (en) 2016-04-15 2022-11-15 Alpine Immune Sciences, Inc. CD80 variant immunomodulatory proteins and uses thereof
US12110339B2 (en) 2016-04-15 2024-10-08 Alpine Immune Sciences, Inc. ICOS ligand variant immunomodulatory proteins and uses thereof
WO2017181148A2 (fr) 2016-04-15 2017-10-19 Alpine Immune Sciences, Inc. Protéines immunomodulatrices à variants du ligand icos et leurs utilisations
US11834490B2 (en) 2016-07-28 2023-12-05 Alpine Immune Sciences, Inc. CD112 variant immunomodulatory proteins and uses thereof
WO2018022946A1 (fr) 2016-07-28 2018-02-01 Alpine Immune Sciences, Inc. Protéines immunomodulatrices à variants de cd155 et leurs utilisations
US11471488B2 (en) 2016-07-28 2022-10-18 Alpine Immune Sciences, Inc. CD155 variant immunomodulatory proteins and uses thereof
WO2018022945A1 (fr) 2016-07-28 2018-02-01 Alpine Immune Sciences, Inc. Protéines immunomodulatrices à variants de cd112 et utilisations associées
WO2018075978A1 (fr) 2016-10-20 2018-04-26 Alpine Immune Sciences, Inc. Protéines immunomodulatrices sécrétables de type variant et thérapie cellulaire utilisant des cellules obtenues par génie génétique
EP3872180A1 (fr) 2016-10-20 2021-09-01 Alpine Immune Sciences, Inc. Protéines immunomodulatrices sécrétables de type variant et thérapie cellulaire utilisant des cellules obtenues par génie génétique
WO2018170023A1 (fr) 2017-03-16 2018-09-20 Alpine Immune Sciences, Inc. Protéines immunomodulatrices à variants pd-l2 et utilisations associées
US11096988B2 (en) 2017-03-16 2021-08-24 Alpine Immune Sciences, Inc. CD80 variant immunomodulatory proteins and uses thereof
US11732022B2 (en) 2017-03-16 2023-08-22 Alpine Immune Sciences, Inc. PD-L2 variant immunomodulatory proteins and uses thereof
US11117950B2 (en) 2017-03-16 2021-09-14 Alpine Immune Sciences, Inc. CD80 variant immunomodulatory proteins and uses thereof
US11117949B2 (en) 2017-03-16 2021-09-14 Alpine Immune Sciences, Inc. CD80 variant immunomodulatory proteins and uses thereof
US11117948B2 (en) 2017-03-16 2021-09-14 Alpine Immune Sciences, Inc. CD80 variant immunomodulatory proteins and uses thereof
US11230588B2 (en) 2017-03-16 2022-01-25 Alpine Immune Sciences, Inc. CD80 variant immunomodulatory proteins and uses thereof
EP4306537A2 (fr) 2017-03-16 2024-01-17 Alpine Immune Sciences, Inc. Protéines immunomodulatrices de variants de pd-l1 et utilisations associées
WO2018170021A1 (fr) 2017-03-16 2018-09-20 Alpine Immune Sciences, Inc. Protéines immunomodulatrices de variants de pd-l1 et utilisations associées
US11639375B2 (en) 2017-03-16 2023-05-02 Alpine Immune Sciences, Inc. CD80 variant immunomodulatory proteins and uses thereof
WO2018170026A2 (fr) 2017-03-16 2018-09-20 Alpine Immune Sciences, Inc. Protéines immunomodulatrices à variants de cd80 et leurs utilisations
EP4219540A2 (fr) 2017-10-10 2023-08-02 Alpine Immune Sciences, Inc. Protéines immunomodulatrices à variants ctla-4 et leurs utilisations
US11753458B2 (en) 2017-10-10 2023-09-12 Alpine Immune Sciences, Inc. CTLA-4 variant immunomodulatory proteins and uses thereof
WO2019074983A1 (fr) 2017-10-10 2019-04-18 Alpine Immune Sciences, Inc. Protéines immunomodulatrices de variants de ctla-4 et leurs utilisations
EP4442268A2 (fr) 2017-10-10 2024-10-09 Alpine Immune Sciences, Inc. Protéines immunomodulatrices à variants ctla-4 et leurs utilisations
US12358969B2 (en) 2017-10-10 2025-07-15 Alpine Immune Sciences, Inc. CTLA-4 variant immunomodulatory proteins and uses thereof
US11613566B2 (en) 2017-10-18 2023-03-28 Alpine Immune Sciences, Inc. Variant ICOS ligand immunomodulatory proteins and related compositions and methods
WO2019079520A2 (fr) 2017-10-18 2019-04-25 Alpine Immune Sciences, Inc. Protéines immunomodulatrices à variants de ligand de icos variant, compositions et méthodes associées
WO2019136179A1 (fr) 2018-01-03 2019-07-11 Alpine Immune Sciences, Inc. Protéines immunomodulatrices multi-domaine et leurs méthodes d'utilisation
US12297253B2 (en) 2018-01-03 2025-05-13 Alpine Immune Sciences, Inc. Multi-domain immunomodulatory proteins and methods of use thereof
WO2019241758A1 (fr) 2018-06-15 2019-12-19 Alpine Immune Sciences, Inc. Protéines immunomodulatrices de variants de pd-l1 et utilisations associées
US12065476B2 (en) 2018-06-15 2024-08-20 Alpine Immune Sciences, Inc. PD-1 variant immunomodulatory proteins and uses thereof
WO2020113141A2 (fr) 2018-11-30 2020-06-04 Alpine Immune Sciences, Inc. Protéines immunomodulatrices à variants cd86 et leurs utilisations

Similar Documents

Publication Publication Date Title
EP3020816A1 (fr) Thérapie génique à médiation bactérienne
Chaturvedi et al. Hypoxia-inducible factor–dependent breast cancer–mesenchymal stem cell bidirectional signaling promotes metastasis
Byrne et al. Bacterial-mediated DNA delivery to tumour associated phagocytic cells
JP7685044B2 (ja) 受容体ｔａｃ技術による膜結合タンパク質の下方制御
KR102190996B1 (ko) Cxcl-1, mcp-1, 또는 timp-1를 발현하는 태반 융모막판막 유래 중간엽 줄기세포를 포함하는 갑상선 안질환을 치료하기 위한 조성물
JP2025023986A (ja) 免疫細胞増殖を促進する方法
KR20220061148A (ko) 세포 노화 조절 방법 및 조성물
KR102604398B1 (ko) 암의 진단 및 치료용 dna 컨스트럭트
EP4294822A1 (fr) Amélioration de la condition métabolique de lymphocytes t pour traiter le cancer
Chen et al. Prolactin enhances T regulatory cell promotion of breast cancer through the long form prolactin receptor
CN108559748B (zh) 一种cd4阳性细胞特异的dna核酸适体及其嵌合体
JP7429046B2 (ja) 腫脹発生抑制型腫瘍溶解性ウイルス
WO2017137461A1 (fr) Vecteur d'expression plasmidique codant pour l'interleukine 12 humaine sous le contrôle transcriptionnel du promoteur p21 et sans résistance aux antibiotiques pour thérapie génique anti-cancéreuse et autres utilisations
TWI823402B (zh) 多特異性單域抗體嵌合抗原受體及t細胞銜接體、核酸、其表達細胞、其用途以及治療癌症之醫藥組合物
Sutherland et al. Expression of the chemokine receptor CCR6 in the Lewis lung carcinoma (LLC) cell line reduces its metastatic potential in vivo
Chen et al. Administration of lipid nanoparticle-encapsulated mRNA encoding CCR7, CXCL16 and GITRL effectively mobilizes antitumor T cell immunity
Zhu et al. Regulation of expression level of fms-like tyrosine kinase-4 is related to osteoclast differentiation
Yu et al. In vitro non-viral lipofectamine delivery of the gene for glial cell line-derived neurotrophic factor to human umbilical cord blood CD34+ cells
KR102253790B1 (ko) 임신성 고혈압 동물모델 및 이의 제조방법
US20220228165A1 (en) DNA Construct for Diagnosing and Treating Cancer
JP6714263B2 (ja) 免疫調節剤及びスクリーニング方法
Zheng et al. Deficiency of Hv1 Proton Channel Enhances Macrophage Antigen Presentation and Anti-Tumor Responses in Glioma
Jeon et al. Controlling tumor progression and recurrence in mice through combined treatment with a PD-L1 inhibitor and a designer Salmonella strain that delivers GM-CSF
CA3190792A1 (fr) Methodes et compositions pour l'evaluation et le traitement du cancer du pancreas
Bühler et al. 1066 A simple method to prepare tumour stem cells from the human breast cancer cell line MDA-MB 231

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

18D Application deemed to be withdrawn

Effective date: 20161119

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN